Ultraviolet light induced cyclobutane pyrimidine dimers (CPD) lesions are the major DNA damage that organis have faced during eons of evolution, therefore, it is conceivable that cells may have developed mechanisms to modify CPD to facilitate its recognition and repair. It has been long recognized that purified UvrABC nuclease, the major repair complex of E. coli, does not excise CPD efficiently; however, the excision can be greatly enhanced by the binding of photolyase to CPD. The extent and rate of repair of CPD by purified nucleotide excision repair (NER) proteins from mammalian cells is also far smaller than suggested by in vivo results. Intriguingly, it has been found that in human cells 80% of the excised CPD contain a disrupted phosphodiester bond (we term this type of cyclobutane pyrimidine dimers "CPD*"). This finding raises the possibility that CPD* lesions are modified CPD lesions and serve as better substrates for NER nuclease in mammalian cells. We have developed a method to construct DNA fragments containing a site- specific CPD* and have found that CPD lesions are indeed much better substrates than CPD not only for the UvrABC nuclease but also for cell- free extracts of human cells. Our findings raise the possibility that CPD* may be the product of preincision process facilitating NER in human cells. To address this question we have developed a method using a photoreactivation mechanism to quantify CPD* in genomic DNA and a method using photoreactivation in combination with ligation-mediated polymerase chain reaction to map the distribution of CPD* at the nucleotide level. Our preliminary results show that a significant amount of CPD in the genomic DNA are converted to CPD* at early incubation time. Moreover, we have confirmed the phenomenon of so called "rodent cell repair paradox" i.e., UV-irradiated rodent cells are able to resume semi-conservative DNA replication to normal levels without repairing 80 % of CPD. These findings led us to hypothesize that conversion of CPD to CPD* is a step that not only facilitates excision repair, but also translesion DNA replication in rodent cells. This proposal aims to test this hypothesis by determining the formation of CPD* in the replicated DNA, the effect of CPD* on DNA replication in both rodent and human cells and their relevancy to the "rodent cell repair paradox", the kinetics and the sequence-specificity of CPD* formation and its repair in mammalian cells, and gene(s) that may be involved in the formation of CPD*. We will also determine the effect of CPD* on the DNA helix structure and construct a molecular model of DNA helix containing CPD* in order to better understand CPD* lesions.